Encapsulation of pharmaceuticals in biodegradable polymer microspheres and nanospheres can prolong the maintenance of therapeutic drug levels relative to administration of the drug itself. Sustained release may be extended up to several months depending on the formulation and the active molecule encapsulated. However many bioactive molecules, and especially proteins, are damaged or made unstable by the procedures required to encapsulate them in the polymeric carriers. Furthermore, the charged, polar nature of many proteins may limit the extent of encapsulation in polymer drug carriers and may lead to rapid loss of a fraction of the encapsulated bioactive molecule when first administered (xe2x80x9cburstxe2x80x9d).
Encapsulation of bioactive molecules in biodegradable polymer delivery systems has been used to stimulate an immune response when administered to a patient (see U.S. Pat. No. 5,942,253 to Gombotz et al.). While this is a desired result in certain cases, it is undesirable when the purpose is delivery of the bioactive molecule for therapeutic purposes. Thus, diminished recognition by the immune system of bioactive molecules delivered using biodegradable polymers would be beneficial in a therapeutic setting.
Bioactive molecules, especially therapeutic proteins (drugs), may be modified with hydrophilic polymers (a process generally known as xe2x80x9cpegylationxe2x80x9d), such as polyethylene glycol, covalently attached to one or more amino acid side chains (see e.g., U.S. Pat. No. 4,179,337 to Davis et al.; U.S. Pat. No. 5,446,090 to Harris; U.S. Pat. No. 5,880,255 to Delgado et al.). While it is known in the art that such attachment may lead to an apparent increase in molecular mass and decreased blood clearance rate for the modified therapeutic protein (see e.g., U.S. Pat. No. 5,320,840 to Camble et al.), the prior art does not teach that diminished immunogenicity can be achieved or that the duration of release from biodegradable polymer drug delivery systems can be extended using pegylated proteins. The prior art does not teach that pegylation can increase the drug loading achievable in a biodegradable drug delivery system relative to the unpegylated drug, nor does it teach that reduced burst of drug is achievable for the pegylated moiety relative to the unpegylated drug.
The present invention provides novel formulations for controlled, prolonged release of bioactive molecules such as therapeutic proteins, peptides and oligonucleotides. The formulations are based on microparticles or nanoparticles formed of the combination of biodegradable, polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and copolymers thereof. Bioactive molecules are coupled to hydrophilic polymers such as polyethylene glycol or polypropylene glycol and then formulated with the solid microparticles or nanoparticles to provide controlled release. The controlled release formulations can be administered by injection, by inhalation, nasally, or orally.
Accordingly, as part of the present invention, it has been discovered that attachment of hydrophilic polymers to bioactive molecules, such as drugs and therapeutic proteins, has several beneficial effects, including providing protection from degradation and denaturation under the conditions of encapsulation in drug carriers. Additionally the amount of modified protein that can be encapsulated is increased relative to the unmodified protein, thus providing a lower total dose of material, benefiting both the patient and producer.
In addition, the present invention is further based on the discovery that immunogenicity of peglyated bioactive molecules encapsulated in biodegradable polymer drug delivery carriers is decreased relative to non-peglyated bioactive molecules in the carriers, particularly when administered by subcutaneous or intramuscular injection or inhalation or mucosal delivery (e.g., oral or nasal delivery). Such diminished immunogenicity is particularly advantageous when biodegradable polymers are used for oral delivery, since this is a typical method for mucosal vaccination.
In another aspect, the present invention is based on the discovery that pegylated proteins, peptides, oligosaccharides and oligonucleotides, which normally are not absorbed from the gastro-intestinal tract, are made bioavailable by administration in biodegradable polymer systems, particularly nanospheres. The term xe2x80x9cbioavailablexe2x80x9d, as used herein, refers to the fraction of bioactive molecule that enters the blood stream following administration to a subject. The controlled release formulations of the invention increase the bioavailability of bioactive molecules and, in particular, the nanosphere formulations described herein when administered orally. For example, blood levels can be maintained for up to several days following a single oral administration of nanosphere encapsulated peglyated bioactive molecule. Additionally the polyethylene glycol chains protect the bioactive molecules from degradation and denaturation in the process of forming the nanospheres, contribute to increased entrapment of active material, and diminish the xe2x80x9cburstxe2x80x9d effect.
Thus, in a preferred embodiment, the invention provides a pharmaceutical composition for controlled, sustained release and increased bioavailability of a bioactive molecule, which includes a polymer (e.g., PEG) conjugated therapeutic agent encapsulated into nanospheres. In a particularly preferred embodiment, the composition is administered orally.
In a preferred embodiment, the bioactive molecule is selected from the group consisting of xcex1-interferon, xcex2-interferon, xcex3-interferon, erythropoietins, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, interleukin 1, interleukin 2, interleukin 3, interleukin 12, asparaginase, adenosine deaminase, insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, leuteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, antidiuretic hormones, thyroid stimulating hormone, endorphins, enkephalins, biphalin, prolactin, monoclonal antibodies, polyclonal antibodies, antisense oligonucleotides, aptamers, therapeutic genes, heparin, low molecular weight heparin and small bioactive molecules.
Accordingly, the compositions of the present invention can be used to improve in vivo delivery of therapeutic bioactive molecules in several respects. In particular, the invention provides the advantages of reduced immunogenicity, increased bioavailability, increased duration, increased stability, decreased burst and controlled, sustained release of bioactive molecules in vivo.
I. Bioactive Molecules
The term xe2x80x9cbioactive moleculexe2x80x9d, as used herein, refers to any therapeutic protein, peptide, polysaccharide, nucleic acid or other biologically active compound for administration to a subject, such as a human or other mammal. Suitable therapeutic proteins for use in the invention include, but are not limited to, interferon-alphas, interferon-betas, interferon-gamma, erythropoetins, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin 1, interleukin 2, interleukin 3, interleukin 12, asparaginase, adenosine deaminase and insulin.
Suitable therapeutic peptides also include hormones, such as ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, luteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, antidiuretic hormones, thyroid stimulating hormone, endorphins, enkephalins, biphalin and prolactin.
Additional suitable therapeutic proteins include monoclonal and polyclonal antibodies, single-chain antibodies, other antibody fragments, analogs and derivatives. Therapeutic polynucleotides, including antisense oligonucleotides, aptamers and therapeutic genes also can be delivered using the methods and compositions of the invention.
Anticoagulant therapeutics, such as heparin and low molecular weight heparin, also can be delivered using the methods and compositions of the invention. Other suitable therapeutic proteins for the use in the invention include small bioactive molecules, such as anticancer drugs, e.g., paclitaxel, taxotere, doxorubicin and daunorubicin, vincristine, cisplatin, carboplatin, camptothecin and camptothecin analogs, antibiotics, antipsychotics, antidepressants, small molecule drugs for diabetes and cardiovascular disease.
II. Conjugation of Bioactive Molecules to Hydrophilic Polymers
The term xe2x80x9chydrophilic polymerxe2x80x9d refers to any water-soluble linear or branched polymer including, but not limited to, polyethylene glycol and polypropylene glycol and similar linear and branched polymers. Preferably, the molecular weight of the polymer ranges from about 500 daltons to about 50,000 daltons. Hydrophilic polymers for use in the invention typically have a reactive group incorporated for attachment to the bioactive molecule of interest through amino, carboxyl, sulfhydryl, phosphate or hydroxyl functions.
Hydrophilic polymers used in the present invention, such as polyethylene glycol, can be prepared according to standard protocols with one end capped as with a methoxy group and the other end activated for facile conjugation to active groups on bioactive molecules. For example, U.S. Pat. No. 6,113,906 describes the use of succinamidyl succinate or carbamate reactive groups on the polyethylene glycol to react with amine groups on proteins. U.S. Pat. No. 5,446,090 describes the use of sulfone derivatives of polyethylene glycol to form stable bonds with sulfhydryl groups of proteins. U.S. Pat. No. 5,880,255 describes the use of tresyl derivatives for reaction at amine groups of proteins to form a simple, stable secondary amine linkage. The entire contents of these patents is incorporated by reference herein. N-hydroxy succinamide also may be incorporated as the reactive group.
III. Controlled Release Formulations for Polymer Conjugated Bioactive Molecules
The term xe2x80x9ccontrolled releasexe2x80x9d refers to control of the rate and/or quantity of bioactive molecules delivered according to the drug delivery formulations of the invention. The controlled release can be continuous or discontinuous, and/or linear or non-linear. This can be accomplished using one or more types of polymer compositions, drug loadings, inclusion of excipients or degradation enhancers, or other modifiers, administered alone, in combination or sequentially to produce the desired effect.
Zero order or linear release is generally construed to mean that the amount of the bioactive molecule released over time remains relatively constant as a function of amount/unit time during the desired time frame. Multi-phasic is generally construed to mean that release occurs in more than one xe2x80x9cburstxe2x80x9d.
A. Microparticles
In one embodiment, the invention employs biodegradable microparticles for controlled release of polymer conjugated bioactive molecules. As used herein, xe2x80x9cmicroparticlesxe2x80x9d refers to particles having a diameter of preferably less than 1.0 mm, and more preferably between 1.0 and 100.0 microns. Microparticles include microspheres, which are typically solid spherical microparticles. Microparticles also include microcapsules, which are spherical microparticles typically having a core of a different polymer, drug, or composition.
Microparticles for use in the present invention can be made using a variety of biodegradable polymers used for controlled release formulations, as are well known in the art. Suitable polymers for example include, but are not limited to, poly(hydroxy acids) including polylactic acid, polyglycolic acid, and copolymers thereof, polyanhydrides, polyorthoesters, and certain types of protein and polysaccharide polymers. The term xe2x80x9cbioerodiblexe2x80x9d or xe2x80x9cbiodegradablexe2x80x9d, as used herein, refer to polymers that dissolve or degrade within a period that is acceptable in the desired application (usually in vivo therapy), typically less than about five years, and more preferably less than about one year, once exposed to a physiological solution of pH between about 6-8 and at a temperature of between about 25xc2x0 C.-38xc2x0 C.
Preferred polymers include poly(hydroxy acids), especially poly(lactic acid-co-glycolic acid) (xe2x80x9cPLGAxe2x80x9d) that degrade by hydrolysis following exposure to the aqueous environment of the body. The polymer is then hydrolyzed to yield lactic and glycolic acid monomers, which are normal byproducts of cellular metabolism. The rate of polymer disintegration can vary from several weeks to periods of greater than one year, depending on several factors including polymer molecular weight, ratio of lactide to glycolide monomers in the polymer chain, and stereoregularity of the monomer subunits (mixtures of L and D stereoisomers disrupt the polymer crystallinity enhancing polymer breakdown). Microspheres may contain blends of two and more biodegradable polymers, of different molecular weight and/or monomer ratio.
Derivatized biodegradable polymers are also suitable for use in the present invention, including hydrophilic polymers attached to PLGA and the like. To form microspheres, in particular, a variety of techniques known in the art can be used. These include, for example, single or double emulsion steps followed by solvent removal. Solvent removal may be accomplished by extraction, evaporation or spray drying among other methods.
In the solvent extraction method, the polymer is dissolved in an organic solvent that is at least partially soluble in the extraction solvent such as water. The bioactive molecule, either in soluble form or dispersed as fine particles, is then added to the polymer solution, and the mixture is dispersed into an aqueous phase that contains a surface-active agent such as poly(vinyl alcohol). The resulting emulsion is added to a larger volume of water where the organic solvent is removed from the polymer/bioactive agent to form hardened microparticles.
In the solvent evaporation method, the polymer is dissolved in a volatile organic solvent. The bioactive molecule, either in soluble form or dispersed as fine particles, is then added to the polymer solution, and the mixture is suspended in an aqueous phase that contains a surface-active agent such as poly(vinyl alcohol). The resulting emulsion is stirred until most of the organic solvent evaporates, leaving solid microspheres.
In the spray drying method, the polymer is dissolved in a suitable solvent, such as methylene chloride (e.g., 0.04 g/ml). A known amount of bioactive molecule (drug) is then suspended (if insoluble) or co-dissolved (if soluble) in the polymer solution. The solution or the dispersion is then spray-dried. Microspheres ranging in diameter between one and ten microns can be obtained with a morphology, which depends on the selection of polymer.
Other known methods, such as phase separation and coacervation, and variations of the above, are known in the art and also may be employed in the present invention.
B. Nanoparticles
In another embodiment, the invention employs biodegradable nanoparticles for controlled release of polymer conjugated bioactive molecules, particularly for oral administration. As used herein, the term xe2x80x9cnanoparticlesxe2x80x9d refers to particles having a diameter of preferably between about 20.0 nanometers and about 2.0 microns, typically between about 100 nanometers and 1.0 micron.
Formulation of nanoparticles can be achieved essentially as described above for microparticles, except that high speed mixing or homogenization is used to reduce the size of the polymer/bioactive agent emulsions to below about 2.0 microns, preferably below about 1.0 micron. For example, suitable techniques for making nanoparticles are described in WO 97/04747, the complete disclosure of which is incorporated by reference herein.